TDNA 500 copies/mL of a second PIK3CA mutation detectable in their baseline ctDNA samp sample, although in lesser quantities than the the tumour mutation (FigureInterestingly, though in lesser quantities than tumour mutation (Figure two). two). Interestingly, all all six patients with PIK3CA-wildtype archival tumour had detectable circulating PIK3CA sufferers with PIK3CA-wildtype archival tumour had detectable circulating PIK3CA m TrkA Storage & Stability mutations in their in their baseline plasma ctDNA. In all participants,quantity of copies of of PIK3 tations baseline plasma ctDNA. In all participants, the the amount of copies PIK3CA mutations in ctDNA fluctuated over the the coursetreatment, with no clear trend mutations in ctDNA fluctuated over course of of treatment, with no clear trend in re in relation to treatment response or duration. A greater peakpeak quantity of mutant PIK3CA alle tion to remedy response or duration. A higher quantity of mutant PIK3CA alleles in ctDNA did not necessarily appear to correlate with using a shorter survival (Figure two). in ctDNA did not necessarily appear to correlate a shorter survival (Figure two).3.4. Serial Tumour Biopsy 3.4. Serial Tumour Biopsy Sequencing Sequencing Two participants had tumour biopsies biopsies post-clinical trial with Two participants had voluntary voluntary tumour pre- and pre- and post-clinical trial with s ficient tumour material exome sequencing (WES) in conjunction with archival tumour sufficient tumour material for wholefor whole exome sequencing (WES) along with archival tumo from initial from initial diagnosis. diagnosis. In both individuals, 20 with the 20 of gene mutations mutations detected, In each individuals, fewer than fewer than somatic the somatic genedetected, which includes includ predicted functional (deleterious) and non-functional have been popular to all predicted functional (deleterious) and non-functional mutations, mutations, were common to 3 timepoints Most mutations mutations had been exceptional to a single or all but not all th three timepoints (Figure S1).(Figure S1). Mostwere one of a kind to one particular or two but nottwothree timepoints in both sufferers reflecting significant temporal genomic heterogeneity. timepoints in both patients reflecting important temporal genomic heterogeneity. In one patient a single patient (patient X), two tumour biopsies had been obtained at pre and the pre a In (patient X), two tumour biopsies were obtained at each and every of the every of post copanlisib plus trastuzumab time points. In the pre-trialthe pre-trial biopsies, 80/98 (81.six ) post copanlisib plus trastuzumab time points. In biopsies, 80/98 (81.six ) somatic mutations had been shared though shared while only of somatic gene mutations gene commatic mutations have been only 10/33 (30.3 ) 10/33 (30.3 ) of somatic had been mutations w mon to the frequent towards the two tumour biopsies p38α list possibly reflecting a lot more intra-tumoural intratwo tumour biopsies taken post-trial, taken post-trial, possibly reflecting far more heterogeneity because the tumour evolves. tumour evolves. moural heterogeneity as theCancers 2021, 13, 1225 Cancers 2021, 13, x9 of 13 10 ofFigure 2. (a) Serial circulating PIK3CA mutant alleles (ctDNA) in individuals with PIK3CA mutation archival tumour (n = = Figure two. (a) Serial circulating PIK3CA mutant alleles (ctDNA) in patients with PIK3CA mutation inin archival tumour (n6); (b)(b) Serial Circulating PIK3CA mutant alleles (ctDNA) in sufferers withno PIK3CA mutation in archival tumour (n = 6); six); Serial Circulating PIK3CA mutant alleles (ctDNA) in patients with no PIK3.